Neisseria meningitidis remains a leading cause of meningitis and rapidly fatal sepsis, usually in otherwise healthy individuals. The regulatory mechanisms of the major virulence factors of this devastating human pathogen are still not well-characterized. The long-term goal of this research is to elucidate the regulatory mechanisms of virulence determinants in meningococcal pathogenesis and to provide a fuller knowledge base for the development of vaccine strategies and therapeutic interventions. Our specific hypothesis is that our recently discovered two-component regulatory system, misR/misS, is a global regulator mediating the expression of meningococcal virulence determinants. We base this hypothesis on our preliminary data of a misR mutant that shows 1) altered expression in several important virulence genes by microarray analysis, 2) autoregulation of the misRS operon, and 3) altered expression of genes participating in iron assimilation. Built on these results, the experimental focus of this proposal is to provide a broad view of the misR regulon and develop a detailed understanding of the molecular regulatory mechanisms of misR on major virulence factors. In Specific Aim I, we will (i) refine and perform additional microarray analyses to better define the MisR-regulated genes, and (ii) determine the direct and indirect nature of MisR regulation on genes identified by microarray analysis. In Specific Aim II, we will (i) further characterize the MisR-MisS protein-protein interaction, (ii) further elucidate the auto-activation mechanism of misR, (iii) expand our understanding of the MisR-DNA interaction giving rise to direct regulation by MisR, and (iv) define a consensus-binding motif of MisR, thereby further defining the direct regulon of the MisRS system. A genome-wide in silico analysis of promoter regions with the derived MisR binding consensus sequence will be conducted to further elucidate the regulatory scope of the MisR/S two-component system. In Specific Aim III, we will focus on the regulatory control of iron assimilation by the MisRS system. Through (i) characterizing the probable iron-dependent phenotypes, (ii) demonstrating the regulatory mechanism of MisR by EMSA and DNase I footprinting and directly probing the relationship between MisR- and Fur- (ferric uptake regulator) dependent regulation and (iii) establishing the indirect regulatory circuitry of MisR in iron-related genes, we will elucidate how MisR modulates the expression of a subset of iron assimilation machinery. Investigations of the mechanisms governing regulation of virulence genes are essential to understanding the pathogenesis of N. meningitidis and are important to the development of new approaches for meningococcal disease prevention and treatment.